This invention relates to tissue sample analysis, and more particularly to the identification of sectioned or cut cells in tissue sections.
During the last thirty years there has been considerable interest in developing techniques for measuring the DNA content of biological cells. These techniques have been applied to the study of cells from tissue or from body fluids of cancer patients. It is generally believed that these tests can provide information useful for (1) diagnosing cancer, (2) determining the prognosis of a diagnosed cancer, and (3) treating the cancer.
DNA content is a useful diagnostic or prognostic parameter in part because of the fundamental principle that the DNA content of normal cells of any given species falls into a well characterized frequency distribution. The characteristic distribution of per cell DNA occurs because virtually all somatic cells, except when preparing for cell division, possess a specific quantity, (called G.sub.0) of DNA in their nuclei. Prior to their division into two daughter cells, non-resting cells synthesize new DNA, increasing their DNA content to twice G.sub.0. Cells having the double, or G.sub.2, amount of DNA eventually undergo mitosis and cytokinesis, dividing into two daughter cells, each with the G.sub.0 amount of DNA. Thus, if many body cells are each measured for their DNA content, and the number of cells for each DNA value measured is plotted against DNA value, a curve with peaks at the DNA values G.sub.0 and G.sub.2 is obtained. The curve also exhibits positive values for DNA content between these two peaks, the values between the peaks being dependent on the rate of synthesis of new DNA. In the ideal measurement system these two peaks are narrow, as there is little biological variation in the G.sub.0 DNA value of any individual's normal cells.
It has been known for the last 50 years that cancer cells may have a resting DNA value different than G.sub.0. The appearance of a population of cells with a DNA value that diverges from the normal is due to the occurrence of one or more abnormal mitotic events. Abnormal mitotic divisions give rise to clones of cells with DNA values above or below G.sub.0. If such a cancer, i.e., a cancer with cells containing an abnormal amount of DNA, is present in a sample of body cells being measured, complex distributions of DNA, with more than the two peaks representing the G.sub.0 and G.sub.2 DNA values, are often found.
During the last 30 years aberrant DNA distributions in biological samples have been studied as a potential marker for the presence of cancer in the patient from whom the sample was taken. During the last 10 years there have been many hundreds of publications showing that there may be a relationship between the prognosis of a patient and consequently how that patient should be treated for his cancer and the distribution of DNA of his cancer cells. In general, it appears that the more aberrant the DNA distribution, the worse the chance of survival of a given patient for many cancers and the more aggressively that patient should be treated.
A number of approaches have attempted to apply these observations in clinically useful ways. A critical review of research in this area as well as an extensive bibliography can be found in L. G. Koss et al, Flow Cytometric Measurements of DNA and Other Cell Components in Human Tumors: A Critical Appraisal, Human Pathology 20, pp. 528-548, 1989, the disclosure of which is hereby incorporated by reference.
Two major approaches have been developed to measure cell constituents such as DNA. In the first, cells from either a body fluid, e.g., blood, or a body cavity are smeared on to a microscope slide and the slide stained such that the constituent of interest, e.g., DNA, of the cells will absorb light at any point on the slide in an amount proportional to the DNA at that point. The user, looking through a microscope scanning instrument, visually finds cells to be measured. For each such cell, the instrument images that illuminated cell using either a high resolution scanning light spot or camera. Light transmitted through the cell for each picture element is added and the sum of these, which is proportional to the DNA of the cell, is read out to the user or recorded in a data base. Since the dye used to measure the cell can be visually observed the user can find cells he believes to be intact cancer cells. The resulting DNA distribution of the sample can be prognostic of the patient's cancer. This procedure uses the human observer to find representative intact cells on isolated cell samples from body fluids or other samples consisting of isolated dispersed cells.
The method described above generally requires dispersed samples, as opposed to tissue sections or slices, in order to segment, i.e., distinguish, neighboring cells (as are found in tissue sections) from one another and to avoid confounding signals from cut or sectioned cells at the cut surfaces of tissue sections.
Several problems associated with the use of dispersed samples limit the value of approaches that use dispersed samples. Because the number of normal cells far exceeds the number of abnormal cells in most samples of either type, the abnormal DNA values, if present, can be obscured by the G.sub.0 values of the normal cells in the tissue and thus not be found. This problem can be addressed in methods where the sample is a tissue section because the user can locate putative cancer cells from morphological characteristics of the tissue section and gather data on such a subset of cells. Thus the use of tissue sections as samples increases the chances of identifying a population of cells with an abnormal G.sub.0 value. Because the information inherent in the morphology of the tissue is lost in dispersed or disrupted samples, this approach is not possible in techniques limited to these sample types. Furthermore, the procedures used to separate cells from one another in the preparation of a sample of dispersed cells (usually enzymatic digestion or mechanical disruption) generate large amounts of cell debris and damaged cells, both of which interfere with analysis.
The number of cells that must be measured in most applications requires the use of automated instruments. Attempts to automate the technique described above have been largely unsatisfactory. The specimens used with automated instruments, e.g., microscope slide scanners, are usually either body fluid cells or tissue that is minced and enzymatically treated to separate individual cells from one another. Thus these efforts generally suffer from the drawbacks discussed above regarding dispersed samples.
The second major approach referred to above has involved the use of flow cytometers. Flow cytometers, which have been commercially available since 1970, are used to measure DNA and other cell constituents in a broad variety of applications. These instruments measure multiple optical properties including fluorescence at different wavelengths and scatter at different angles, of cells flowing in a capillary past a light excitation whose source can be a laser or arc lamp. The cells analyzed are dispersed and stained to produce fluorescence proportional to one or more cellular constituents of interest. Flow cytometers operate automatically at measurement rates of 1000's of cells per second, provided that samples of isolated cells are available in liquid suspension.
Although flow cytometers measuring per cell DNA have been widely used for cancer prognosis, there is considerable criticism of the accuracy of the results. All of the problems associated with the measurement and interpretation of data gathered with non-flow instruments, e.g., microscope slide scanners, are present when flow cytometers are used. With a flow cytometer, which uses dispersed samples, it is impractical or impossible to obtain DNA distributions on only the cancer cells in a specimen, thus it is impossible or impractical to isolate data representative of just the cancerous part of the tissue specimen. As in the previously discussed techniques, the vast number of normal G.sub.0 cells may obscure the measurement of other cell populations. Since the cells must flow through a capillary in single file, the sample must be traumatically treated to yield suspensions of single nuclei or cells. These samples are contaminated with debris and damaged cells that can interfere with subsequent data analysis.